Superoxide dismutases and their applications as oxidation inhibitors

ABSTRACT

The invention relates to the application of superoxide dismutases for inhibiting the degradation of auto-oxidizable substances. An effective amount, preferably from 1 to 200 units per milliliter or per gram of substance, of at least one superoxide dismutase is associated with the substances to be protected. Application notably to preserving foodstuffs, bacteria, viruses and other oxidizable substances.

United States Patent 91 Michelson et al.

[451 Nov. 18, 1975 1 SUPEROXIDE DISMUTASES AND THEIR APPLICATIONS AS OXIDATION INHIBITORS [75] Inventors: Adolf Michael Michelson,

Chatenay-Malabry; Jacques Monod, 1 Paris, both of France [73] Assignee: Agence Nationale de Valorisation de la Recherche (ANVAR),

'Neuilly-sur-Seine, France 22] Filed: Apr. 16,1975

[21] Appl. No.: 461,426

[30] Foreign Application Priority Data McCord et al., superoxide Dismutase, Journal of Biological Chemistry 244 (1969); Pp. 6049-6055.

Webb et al., Fundamentals of Dairy Chemistry, Oxidative Deterioration in Dairy Products, Avi Publishing Co. (1965), PP. 204-207.

Primary Examiner-A. Louis Monacell Assistant Examiner-R. B. Penland Attorney, Agent, or Firm-Hammond & Littell [57 ABSTRACT The invention relates to the application of superoxide dismutases for inhibiting the degradation of autooxidizable substances.

An effective amount, preferably from 1 to 200 units per milliliter or per gram of substance, of at least one superoxide dismutase is associated with the substances to be protected.

Application notably to preserving foodstuffs, bacteria,

- viruses and other oxidizable substances.

16 Claims, No Drawings SUPEROXIDE DISMUTASES AND THEIR APPLICATIONS AS OXIDATION INHIBITORS This invention relates to the application of superoxidc dismutases as oxidation inhibitors, notably as oxidation inhibitors for foodstuffs and other compounds or oxidizable media.

It has now been discovered that superoxide dismutase enzymes of any origin are remarkably effective as oxidation inhibitors for oxidizable substances, and notably as oxidation inhibitors for lipid, protein or lipiprotein foodstuffs, as well as other. substances and oxidizable media.

The object of the invention according to the present application is the application of superoxide dismutase enzymes as oxidation inhibitors for oxidizable substances, notably foodstuffs, antioxidants used in the foodstuff industry, and bacteria and other oxidizable media.

In another aspect, the invention relates to a process for controlling the oxidation of oxidizable substances, characterized in that an effective amount of at least one superoxide dismutase enzyme is associated with said substances.

Within the meaning of thepresent invention, oxidizable substance is taken to mean any substance liable to be degraded by oxidization involving superoxide ions. i

The superoxide dismutases which have been found to be effective are, for example, those prepared according to the copending application, filedon 'even date in the name of Adolf Michael Michelson, and entitled New superoxide dismutase and its application Ser. No. 461,379 as an oxidation inhibitor, from marine bacterial strains such as, for i'nstance,'strains of Photobacterium phosphoreum, Photobacterium leiognathi or Photobacterium sepia, among ,others; but can also be any other superoxide dismutases such as, an non-limiting examples, those extracted from Escherichia coli, fungi such as Pleurotus oleariiis' or those extracted from blood, notably erythrocupreines. Among said different strains may be mentioned strains of Photobqcter'ium flphosphoreum No. ATCC 11,040, PhOtObdCtertlllm .No. ATCC 15,709, Escherichia coli No. ATCC 15,224 and Pleurotusolearius'Gillet (Cryptogamy Laboratory, Paris).

The remarkable anti-oxidization effect of said superoxide dismutases has been demonstrated on mushrooms, apples and potatoes, among other foodstuffs.

of the'inhibition of lipo-protein auto-oxidation, tests were effected on the auto-oxidation inhibiting activity provided according to the invention by dismutases on lysine RNA-t yeast ligase, a model representing all 'classes of lipo-proteins. Similarly, as the model of the generality of nucleo-pr'oteins, the bacteriophage R 17, which is a ribonucleoprotein was used. In another connection, oxidation of the pancreatic ribonuclease enzyme was tested as a model for foodstuffs of a strictly protein character.

2 In another connection, the efficacy of superoxide dismutases for protecting bacteria exposed to ultra-violet radiation from oxidization was also established, as also for the protection of a medium, known as the Jeffries medium, containing sodium tetrathionate and active for the enrichment of Salmonella.

As was stated above, the superoxide dismustase enzyme used according to this invention can be extracted from any suitable source, such as: erythrocupreine, Escherichia coli, fungi such as Pleurotus olearius, marine bacterial strains such as Photobacterium Phosphoreum, Photobacterium Ieiognatlzi and Photobacterium sepia, and the like. Whatever the source of the enzyme, assessment of its efficacy in the application in question remains the same, as it is measured in absolute terms: the activity of the enzyme is determined by inhibition of the chemioluminescence of luminol, a reaction produced by the enzyme system hypoxanthine/xanthine oxidase/oxygen; each time said reaction was effected, 1 ml 0.33 X l0 M hypoxanthine was injected into a mixture of 0.3 ml 10*"M luminol, 0.3 ml of l M, pH 9 NaOl-I glycine buffer, 0.3 ml lO M EDTA, 1.8 ml water and 0.005 ml of a l mg/ml xanthine oxidase solution. The experimental device used to determine light intensities (1) without, and (2) following, inhibition by means of superoxide dismutase was that described in the copending application, and comprising a silver-lined cuvette adapted to receive the mixture to be tested and positioned in front of a photomultiplier. The reaction was initiated by injecting the substrate into the cuvette, following which emission of a photon flux occurred which, in response to the photomultiplier, gave rise to a current the intensity of which was measured with a picoamperemeter and recorded. If a suitable amount of the superoxide dismutase to be determined was introduced into the reaction mixture, prior to reaction initiation, said light emission was inibited. The unit of activity selected corresponding arbitrarily to the amount of enzyme inducing a 50% reduction in the maximum light intensity in the absence of enzyme inhibition. Said unit is therefore independent of the enzyme source and the superoxide dismutase enzyme used.

In certain cases, it may be advantageous to activate oxidation of the materials to assess the efficacy of the superoxide dismutases in the application in question over areasonable period of time. For this, the operation is in practice effected either with flavines reduced by 265 my. irradiation, or by means of an enzyme system, such as a xanthine oxidase/hypoxanthine/oxygen system.

When oxidation is accelerated by reducedflavines, a solution of 10M flavine mononucleotide (FMN), a 10"M EDTA and IO M, pH 7.0 phosphate buffer was irradiated in a quartz cuvette placed in a Zeiss spectrofluorimeter provided with a M365 filter, photoreduction, which occurs rapidly, is followed by observing the decrease in the intensity of fluorescence emitted at 530 At the end of reduction, the solutions are recovered and stirred vigorously in the presence of air, this resulting in their complete reoxidation, which produces O; superoxide ions. It should be noted that said flavine reduction-reoxidation cycle can be repeated several times without the structure of the mononucleotide being impaired. As a variant, irradiation at 365 my. can be effected with a BIOO A lampproduced by Ultraviolet Products, lnc., on constantly stirred solutions such as those described hereinabove.

When it is desired to accelerate oxidation by the hypoxanthine/xanthine oxidase/oxygen enzyme system, it is advantageous to use a solution containing 0.3 ml lO' M hypoxanthine, 0.03m1 1M, pH 7.8 phosphate buffer, 0.3 ml *M EDTA, 2.1 ml water and 0.05 ml of a I mg/ml xanthine oxidase solution.

Consequently, from still another point of view, the invention relates to foodstuff compositions, compositions obtained from strains of bacteria or viruses or culture mediums comprising, in association with the said foodstuffs, bacteria, viruses or other constituents of the medium, an effective amount of a superoxide dismutase enzyme.

It is clear for those skilled in the art that the amounts of superoxide dismutase enzymes to be associated with the substance to be protected are not critical, and any man of the art is capable of determining the most suitable amounts in each specific case. Routine trials enable the efficacy of the protection provided by the enzyme to be determined, and the amount of enzyme to be modified accordingly if necessary, using for this purpose tests of activity similar to those described above.

It can however be indicated that, as an example, amounts of superoxide dismutase enzyme in the range of l to 100 units per milliliter or per gramme are particularly suited for the protection of auto-oxidable substances as they have been defined hereinabove.

As has been demonstrated in the above referred cop ending application, superoxide dismutases effectively protect lipids and anti-oxidants and other preservatives usually used in the food industry, by very strongly inhibiting the reactions connected with the production of the O superoxide ion. It was notably established that the auto-oxidation of unsaturated lipids obtained from fish is very strongly inhibited by superoxide dismutases. Furthermore, other trials have shown that superoxide dismutases have a protective effect on the auto-oxidation of certain anti-oxidants, notably the anti-oxidants used for preserving foodstuffs, such as pyrogallol or ascorbic acid.

The invention is further illustrated by the following examples, which are illustrative of this invention rather than restrictive of its scope.

EXAM PLE l 5 g of fresh mushrooms were used; these were thinly sliced and the coloured grills removed as they might have affected the colour of the medium andmade it difficult to access the results.

The mushrooms so prepared were divided between several 100 ml beakers and 50 ml of a pH 7.8 (0.01M phosphate buffer) buffered solution containing 0.02% ascorbic acid was added.

50 units of superoxide dismutase of Photobacterium leiognathi, strain No. ATCC 25 ,5 21 was added to one of said beakers.

Each beaker was covered with a double sheet of paper and left to stand at laboratory temperature.

40 hours later, the optical density of the supernatent in each of the beakers was determined at 600 mp. the optical density found made it possible to deduce an average increase of 0.173 in the optical density for the controls, whereas the increase in optical density was only 0.092 for the supernatent comprising the superoxide dismutase. Oxidation in the beaker so treated was therefore 47% lower than it was in the control beakers.

EXAMPLE 2 EXAMPLE 3 Slices of potato were placed in beakers each containing 3units of superoxide dismutase of Photobacterium leiognathi strain n) ATCC 25,521 per ml in a pH 7.8

. phosphate buffer, they were removed a few minutes later, placed in Petri dishes and left to stand. Four days late the treated slices appeared to be markedly less oxidized than identical slices subjected to the same treatment but without the addition of superoxide dismutase.

EXAMPLE 4 It is known that certain RNA-t yeast ligases are lipoproteins in which the, enzyme activity is dependent upon the integrity of the lipid portion. A material is therefore available the breakdown of which can be objectively determined by a simple assessment of its enzyme activity and which, owing to its essentially lipoprotein composition, can be taken as a model for all lipo-protein foodstuffs.

The ligases used were extracted from yeast cells (Saccharomyces cerevisiae). The following procedure was used to prepare them: strains of Saccharamyces cerevisiae No. ATCC 9841 were cultured in a medium containing 30g glucose and 5g yeast extract, the culture being left to develop until the middle of the logarithmic phase of growth. Centrifugation was then effected, after which the cellswere washed and ground in a device known as a French Press; 67g of yeast were thus treated with 67 ml buffer (consisting of 0.01 M tris pH 8, 0.01M MgCl 1 ml EDTA, 10% glycerol and 2mM phenyl-methyl-sulfonium fluoride). Following three grinding steps in the device known as a French Press, the solution was cenrifuged for 30 minutes at 15,000 revolutions/minute. The supernatent was reduced and centrifuged again for 2 hours at 50,000 revolutions/minute. The supernatent from this last centrifugation was purified on a 2cm X 36 cm DEAE-cellulose DE-52 column comprising g of cellulose, the said column being equilibrated with 0.02M, pH 7.5 potassium phosphate buffer, 0.02 M mercaptoethanol, lmM MgCl and 10% glycerol. The said supernatent was poured into the column and washed with 370 ml of the same buffer. The ligases were then eluted with a 0.25 M, pH 6.5 potassium phosphate buffer, 0.02 M mercaptoethanol, 1 mM of MgCl and 10% glycerol.

The optical densities of the fractions recovered at the bottom of the column were determined and activity was tested with lysine and the other amino acids (levels of activity were given in table I below). Each extract was then concentrated by ultrafiltration to a protein concentration of 10 mg/ml and determinations were effected by measuring the charge of RNA-t. ul of the incubation medium were used, containing 50 mM N-morpho-lino-3-propane sulphuric acid (pH 6.5). 10

mM MgCl 2mM ATP (adenosine-triphosphoric 5 acid), 40 pM (picomoles) of carbon l4-labelled lysine and 10 units of total/yeast RNA-t with 260 mp. optical density.

After incubation for a predetermined period of time, 50 pl of the reaction mixture was deposited on Whatmann DE-8l paper and washed for one and one half hours with 8.7% acetic acid and 2.5% formic acid. The

paper so washed was then dried and, on the spots formed, counting was effected using a scintillator mixture containing toluene.

To determine the efficacy of the protection of lysine RNA-t yeast ligase from oxidation obtained with a superoxide dismutase, a solution of 1.37 mg lipoproteins in 1 ml (0.5M) pH 7.5 phosphate buffer was formed and either 0.1% ascorbic acid, or 90 units of the superoxide dismutase of Ph tob cteri gnathi, strain No. ATCC 25,521 was added; in each case, the solutions were left to stand at 4C, aliquot parts were withdrawn at various times (see table 1 below) and enzyme activity was determined as previously described.

Similar results were obtained using the same number of units of superoxide dismutases obtained from Pleuratus olearius Gillet, Escherichia coli strain No. ATCC 15,224 or erythrocupreine.

EXAMPLE 5 In order to determine the protection from oxidation provided to bacteria by superoxide dismutase, luminescent bacteria of Photobacterium leiognathi strain No. ATCC 25,521 were used, the oxidation of which was artificially accelerated by photoreduction of FMN in order to render the phenomenon of protection perceptible 'over a shorter period of time. The bacteria were cultured at 28C on amedium comprising 8g of nutrient broth, 10g NaCl, 15g agar-agar, and water to make up to 1000 ml, the pH of this medium being adjusted to 7. Cells were counted' by spreading 0.1 ml ofa bacterial solution diluted with physiological salt solution in Petri dishes maintained at 25C and containing a medium consisting of 8g nutrient broth, 10g NaCl, 15g agaragar, and water'to make up to 1000 ml,the pH of said medium being adjusted to 7.

A culture of 100 ml Photobacterium leiognathi was centrifuged in exponential development (their optical density at 600mg was 0.3) at a velocity of 10,000 revolutions/minute, for 10 minutes at 3C. The centrifugation residue was dispersed in 100 ml physiological salt solution. Said bacterial suspension was diluted 10 times in 5 X l' M FMN and 3% NaCl (respective proportions) to a final volume of ml.

Bacteria was irradiated at 25C at 365 mp, under a B100 A lamp and with constant stirring. After predetermined durations of exposure to the lamp, aliquot parts were withdrawn and the cells were counted, after which they had been further diluted with physiological salt solution. Whereas prior to irradiation l X 10 cells 6 were present, after determined durations of irradiation the number of cells/ml counted are given in table 11 below.

TABLE II Duration of Control Bacteria with 13.5

exposure units/ml superoxide dismutase (in 10*M pH7 phosphate buffer) 30 4 X l0"" 7 X 10' 60 5 X 10" 3.9 X10 120 8X10" 6X10 EXAMPLE 6 Using the same procedure as in example 5, bacteria of Photobacterium leiognathi, strain No. ATCC 25,521 were put in suspension in a physiological salt' been added 53 units per ml of superoxide dismutase (SOD) of Pleurvtus olearius Gillet, the results obtained being as follows:

10 cells/ml 10 cells/ ml Non-irradiated bacteria lrridiated bacteria (control) Irradiated bacteria (with 53 units/ml of Pleurotus olearius SOD) 5.0 X 10 cells/ml EXAMPLE 7 A trial was conducted to preserve a bacteriophage, which is a nucleoprotein and will in the following be referred to as bacteriophage R 17. Said bacteriophage was preserved and diluted in a solution comprising 6g NaHPO. 3g KH PO 0.5 g NaCl, lg NH Cl, ml water and 10ml 0.01M CaCl 0.1 ml of said bacteriophage solution was incubated for ten minutes at 38C with 0.2 ml of a solution of Escherichia coli strain n ATCC 15,224, 10g trypticase, 5g yeast extract, 10g NaCl and 1000 ml water, and at an optical density of 0.5 at 650 my. with 3 ml of soft gelose suspension (consisting of 8g agar, 10g trypticase, lg yeast extract, 8g NaCl, 1000 ml water, 2 ml 1 M of CaCl and 5 ml 20% glucose) which was maintained at 45C. The mixture was poured into Petri dishes containing 20 ml hard gelose (consisting of 12 g agar, 10g trypticase, 1g yeast extract, 8g NaCl, 1000 ml water, 2 ml 1 M CaCl and 5 ml of 20% glucose); and the Petri dishes were placed at a temperature of 37C.

In order to accelerate the natural oxidation of bacteriophage R 17, photoreduction of flavin mononucleotide was used: the bacteriophage solution was diluted with 10 times its volume of a solution of 10' MFMN, l0 M EDTA and 10 M pH 7 phosphate buffer, photoreduction was effected with a spectrofluorometer, the final volume of the solution being 0.8 ml.

With a solution initially containing 4.1 X 10 infective particles/ml at moment t 0, the results given in table [11 below were obtained.

TABLE III PARTlCLES/ ml Number of succes- Control trial sive photoreduc- (comprising 0.05 mg with 13.5 tion operations dismutase inactivunits/ml ated at pH 3 (A) dismutase Protection (B) (B) relation (A) 41x10" 4.1 x10" 3 49 x1 1 7.5 10 1.53 6 5 x 1.5 10 3.00 9 5 x10 2 10 4.00

EXAMPLE 8 TABLE V-continued t 7r residual activity Proceeding as in the prev ous example, except that, Comm. Comm with Dismumse in order to accelerate ox1dat1on of the bactenophage R Number f (without dismurase (Q05 mg) 6,7,5 17, 0.3 ml of the latter was put to incubate with a cycle5 dismutase) inactivated at P 3 unItS/ml hypoxanthine/xanthine oxidase/oxygen enzyme system, 3 67 r 56 39 the volume of the incubation system being 3 ml. 8 g; g; 2;

After 15 minutes, the system was again treated by the addition of 0.05 ml xanthine oxidase and 0.3 ml 10*M hypoxanthine.

With, at time zero, 5.0 X 10" particles/ml, the results EXAMPLE 10 given in table IV below were obtained in the absence and in the presence of 200 units/ml of superoxide dismutase obtained from Photobacterium sepia strain n ATCC 15,709. Similar results were obtained with superoxide dismutases of Pleurotus olearius Gillet or ery- A superoxide dismutase, enzyme, strain Phorobacterz'um leiognathi No. ATCC 25,521, was used in an attempt to obtain improved preservation of the medium known as the Jeffries medium, containing sodium tetrathionate.

throcupreine.

TABLE IV Particles/ml Number of succes- Without With dismutase Mortality Mortality sive enzyme dismutase (200 units) "/1 control (with disoperations (control) mutase) 0 5.0 X 10 5.0 X10" l. 3.5 X10 5 X10 3O 0 2 2 x 10 3 x 10 60 40 Said medium is a Salmonella enrichin medium and EXAMPLE 9 g 1s used conventionally as follows: a sample containing The protection of pancreatic ribonuclease from oxill amounts f S lmon lla and large amounts of dation by the u Of superox de diSmutaSeS Was tested Escherichia coli is introduced into such a sodium tetraby realizing oxidation by means of photoreduction of rhi r di Af 24 hours i an o at 37C, FMN to rend r the ph n m n pe ep i e 8 one drop of said culture was seeded on agelose culture more Suitable period. For this purp e. a l n of in a Petri dish. After 24 hours incubation at 37C, a pancreatic ribonclease solution in 10 2M, very large number 0f Salmonella colonies were 1 pH 7 ph sphate buffer an 0 M EDTA) was diluted served in said culture but absolutely no Escherichia coli. in 10 tim s it ol m f a Solution consisting of It was thus confirmed that the said sodium tetrathio- FMN, 10""M EDTA and I M, P 7 ph ph nate medium inhibits Escherichia coli but encourages buffer, the final volume being 0.8 ml, the pancreatic rih d l t f S l n ll Ho ver, it wa l o bonuclease protein was thus subjected to several phoconfirmed that said medium can only really be used for toreduction cycles, in the absence, and in the presence a maximum i d f thr k f th tim it w of 67.5 units/ml superoxide dismutase obtained from 5 prepared, d even, i ll f a maximum f tw Pleurotus oleari's Gillet or in the presence of the same k F r this reason it has only been possible up till dismutase, denatured and inacti e at P 3- now to produce small batches of such a medium. mak- The activity of the ri nu l as nzym was detefing it difficult to keep a check on stocks and thus inmined using a spectrophotometer, following the increasing costs,

crease Of the optical density at 280mg. Of a ribonucleic To carry out an experiment on the protection of Jefacid solution such as described above. The results obf i di f id io 100 be wer tak n tained are given in table V below: from a same batch of said sodium tetrathionate me- TABLE v dium; 2 ml of a Photobacterium leiognathi superoxide dismutase solution containing 15 units/ml were added iesidiia' activity. to 11 of said tubes which each contained 20 ml of the Control Control with Dismutase Number of (without dismutase 0.05 mg) 67.5 medium, all the other tubes bemg considered as con- Cycles dismutase) inactivated P 3 trols. All tubes were kept together at a temperature 0 100 100 100 of+ 4.0 "C.

Analyses were conducted. to determine .the preservation of the media at a rate of one analysis per week for 7 weeks and then only one analysis every second 'week. For each of said analyses one tubc containing the .enzyme was compared with: g

1 control tube for the first three tests 2 control tubes for the 4th and th tests 3 control tubes for the last five tests.

The procedure described above at the beginning of this example was used for analysis. I

The results are given' in table Vl below.

+ satisfactory result: pure Salmonella culture bad results: ('01! culture 0 bud results: no culture It is immediately obvious from this table that the contents of tubes into which superoxide dismutase enzymes had been introduced retained all their properties. The control tube gave satisfaction in the first three tests, which corresponds to the duration of validity considered up to now to be normal for commercial batches of sodium tetrathionate. The following tests showed that results were irregular for the control tubes, which makes it possible to consider the product as a commercial proposition It is therefore seen that a superoxide dismutase enzyme solution obtained from Photobacte- 'rium leiognathi strain No. ATCC 25,521added at a rate of 2 ml per tube of 20ml sodium tetrathionate lengthened the latters resistance to oxidation by at 1 least weeks, and even longer.

EXAMPLE l Preserving potatoes A 250 of peeled potatoes are cooked in water at 100C for 30 minutes. 60ml of water is added and they are'treated in a mixer until a homogeneous mass is ob- 'tained. Four 50 g samples are placed in beakers and 'ml water is added and thoroughly mixed with the mash.

Beaker Pl Control 1 P2 50 units of superoxide dismutase l0 units/g) P. Ieiognalhi are added and mixed Control plus 0.5 ml 2.07: ascorbic acid(final concentration 0.0271 ascorbic acid) As P3 but 500 units of superoxide dismutase are also added.

All the samples are then freeze-dried and left to stand at ambient temperature.

Potato flakes were used containing mg/kg BHT (butylated hydroxy toluene) and BHA (butylated hydroxyanisole) and glycerol monostearate (1%).

45 g of the aforesaid flakes were added to 250 ml boiling water, the mixture was left for 2 minutes and Ml M2 Control 7 500 units superoxide dismutase (l() units/g mash) added and well mixed.

The two samples are then freeze-dried and the pow- O der left to stand at ambient temperature.

Two months later samples A and B were compared by six persons. The odour of the sample containing superoxide dismutase was different from that of the control, less acid and. more pleasant.

Preserving carrots Exactly the same procedure was used as that described for potatoes, the carrots being cooked in water and the same amount of superoxide dismutase/g being added.

The odour of the two samples was compared two weeks later by six persons. The odour of the sample containing a peroxide dismutase differed from that of the control and was less acid and more pleasant.

' What we claim is:

l. A process to control auto-oxidation of substances liable to be degraded by oxidation with superoxide ions, wherein an anti-oxidation effective amount of at least one superoxide dismutase enzyme is added to said substances.

2; A process according to claim 1, wherein the said substances are selected from the group consisting of lipid, protein, nucleoprotein or lipo-protein foodstuffs.

3. A process according to claim 1, wherein the said substances are bacteria or viruses.

4. A process according to claim 1, wherein the said substances are culture mediums used for culturing cells.

' 5. A process according to claim 1, wherein the superoxide dismutase is incorporated in the said substance at rates of from 1 to 200 units of superoxide dismutase per milliliter or per gram of substance.

6. A process according to claim 1, wherein the superoxide dismutase enzyme is selected from those extracted from marine bacterial strains, from Escherichia coli, fungi orthose extracted from blood, andnotably erythrocupreines. 1

.7. A process according to claim 6, wherein the said marine strains are strains of Photobacterium Ieiognathi, Photobacterium phosphoreum or Photobacterium sepia, notably Photobacterium leiognathi strain No. ATCC 25,521, Photobacterium phosphoreum strain No. ATCC 11,040 or Photobacterium sepia strain No. ATCC 15,709.

8. A composition consisting essentially of substances liable to be degraded by oxidation involving superoxide ions and an anti-oxidation effective amount of at least one superoxide dismutase enzyme to protect the said substances from oxidation or to reduce their oxidation.

9. A composition according to claim 8, wherein the said substances are selected from the group consisting of lipid, protein, nucleoprotein, or lipoproteinfoodstuffs. 1

10. A composition according to claim 8, wherein the said substances are bacteria or viruses.

11. A composition according to claim 8 wherein the said substances are culture mediums used for culturing cells.

12. A composition according to claim 8, which comprises a protein composition and, in association with the protein liable to undergo oxidation, an effective amount of at least one superoxide dismutase enzyme.

13. A composition according to claim 8, wherein the superoxide dismutase is incorporated in the said substance'at a rate of 1 to 200 units of superoxide dismutase enzyme per milliliter or per gram of substance.

14. A composition according to claim 8, wherein the superoxide dismutase enzyme is selected from those extracted from strains of marine bacteria, Escherichia coli, fungi, or those extracted from blood, notably erythrocupreines.

15. A composition according to claim 14, wherein the said marine strains are strains of Photobacterium leiognathi, Photobacterium phosphereum or Photobacte- 

1. A PROCESS TO CONTROL AUTO-OXIDATION OF SUBSTANCES LIABLE TO BE DEGRADED BY OXIDATION WITH SUPEROXIDE IONS, WHEREIN AN ANTI-OXIDATION EFFECTIVE AMOUNT OF AT LEAST ONE SUPEROXIDE DISMUTASE ENZYME IS ADDED TO SAID SUBSTANCES.
 2. A process according to claim 1, wherein the said substances are selected from the group consisting of lipid, protein, nucleoprotein or lipo-protein foodstuffs.
 3. A process according to claim 1, wherein the said substances are bacteria or viruses.
 4. A process according to claim 1, wherein the said substances are culture mediums used for culturing cells.
 5. A process according to claim 1, wherein the superoxide dismutase is incorporated in the said substance at rates of from 1 to 200 units of superoxide dismutase per milliliter or per gram of substance.
 6. A process according to claim 1, wherein the superoxide dismutase enzyme is selected from those extracted from marine bacterial strains, from Escherichia coli, fungi or those extracted from blood, and notably erythrocupreines.
 7. A process according to claim 6, wherein the said marine strains are strains of Photobacterium leiognathi, Photobacterium phosphoreum or Photobacterium sepia, notably Photobacterium leiognathi strain n* ATCC 25,521, Photobacterium phosphoreum strain n* ATCC 11,040 or Photobacterium sepia strain n* ATCC 15,
 709. 8. A composition consisting essentially of substances liable to be degraded by oxidation involving superoxide ions and an anti-oxidation effective amount of at least one superoxide dismutase enzyme to protect the said substances from oxidation or to reduce their oxidation.
 9. A composition according to claim 8, wherein the said substances are selected from the group consisting of lipid, protein, nucleoprotein, or lipoprotein foodstuffs.
 10. A composition according to claim 8, wherein the said substances are bacteria or viruses.
 11. A composition according to claim 8 wherein the said substances are culture mediums used for culturing cells.
 12. A composition according to claim 8, which comprises a protein composition and, in association with the protein liable to undergo oxidation, an effective amount of at least one superoxide dismutase enzyme.
 13. A composition according to claim 8, wherein the superoxide dismutase is incorporated in the said substance at a rate of 1 to 200 units of superoxide dismutase enzyme per milliliter or per gram of substance.
 14. A composition according to claim 8, wherein the superoxide dismutase enzyme is selected from those extracted from strains of marine bacteria, Escherichia coli, fungi, or those extracted from blood, notably erythrocupreines.
 15. A composition according to claim 14, wherein the said marine strains are strains of Photobacterium leiognathi, Photobacterium phosphereum or Photobacterium sepia, notably Photobacterium leiognathi strain n* ATCC 25,521, Photobacterium phosphoreumstrain no* ATCC 11,040 or Photobacterium sepia strain no* ATCC 15,709.
 16. A composition consisting essentially of substances liable to be degraded by oxidation with superoxide ions selected from the group consisting of lipid, protein, nucleoprotein and lipoprotein foodstuffs, bacteria, viruses and culture mediums for culturing cells and an anti-oxidation effective amount of at least one superoxide dismustase enzyme extracted from Escherichia coli, fungi, blood, Photobacterium leiognathi, Photobacterium phosphoreum and Photobacterium sepia to prevent or reduce oxidation. 